Long-lasting aqueous dispersions or suspensions of pressure-resistant gas-filled microvesicles and methods for the preparation thereof

ABSTRACT

One can impart outstanding resistance against collapse under pressure to gas-filled microvesicle used as contrast agents in ultrasonic echography by using as fillers gases whose solubility in water, expressed in liter of gas by liter of water under standard conditions, divided by the square root of the molecular weight does not exceed 0.003.

This application is a division of application Ser. No. 08/380,588, filedJan. 30, 1995, now U.S. Pat. No. 5,578,292, which is a division ofapplication Ser. No. 07/991,237, filed Dec. 16, 1992, now U.S. Pat. No.5,413,774, which claims the benefit of EP92810046.0, filed Jan. 24,1992. This application is also a continuation-in-part of applicationSer. No. 08/456,385, filed Jun. 1, 1995, now U.S. Pat. No. 5,658,551,which is a division of application Ser. No. 08/315,347, filed Sep. 30,1994, now U.S. Pat. No. 5,531,980, which is a division of applicationSer. No. 08/128,540, filed Sep. 29, 1993, now U.S. Pat. No. 5,380,519,which is a division of application Ser. No. 07/775,989, filed Nov. 20,1991, now U.S. Pat. No. 5,271,928, which is the national stageapplication of PCT/EP91/00620, filed Apr. 2, 1991, which claims thebenefit of EP90810262.7, filed Apr. 2, 1990. This application is also acontinuation-in-part of application Ser. No. 08/288,550, filed Aug. 10,1994, now U.S. Pat. No. 5,711,933, which is a division of applicationSer. No. 08/033,435, filed Mar. 18, 1993 and now abandoned, which is acontinuation of application Ser. No. 07/695,343, filed May 3, 1991 andnow abandoned, which claims the benefit of European Patent No.90810367.4, filed May 18, 1990.

TECHNICAL FIELD

The present invention concerns stable dispersions or compositions of gasfilled microvesicles in aqueous carrier liquids. These dispersions aregenerally usable for most kinds of applications requiring gaseshomogeneously dispersed in liquids. One notable application for suchdispersions is to be injected into living beings, for instance forultrasonic echography and other medical applications. The invention alsoconcerns the methods for making the foregoing compositions includingsome materials involved in the preparations. for instancepressure-resistant gas-filled microbubbles, microcapsules andmicroballoons.

BACKGROUND OF INVENTION

It is well known that microbodies or microglobules of air or gas(defined here as microvesicles), e.g. microbubbles or microballoons,suspended in a liquid are exceptionally efficient ultrasound reflectorsfor echography. In this disclosure the term of “microbubble”specifically designates hollow spheres or globules, filled with air or agas, in suspension in a liquid which generally result from theintroduction therein of air or gas in divided form, the liquidpreferably also containing surfactants or tensides to control thesurface properties and the stability of the bubbles. The term of“microcapsule” or “microballoon” designates preferably air or gas-filledbodies with a material boundary or envelope, i.e. a polymer membranewall. Both microbubbles and microballoons are useful as ultrasoniccontrast agents. For instance injecting into the bloodstream of livingbodies suspensions of air-filled microbubbles or microballoons (in therange of 0.5 to 10 μm) in a carrier liquid will strongly reinforceultrasonic echography imaging, thus aiding in the visualization ofinternal organs. Imaging of vessels and internal organs can stronglyhelp in medical diagnosis, for instance for the detection ofcardiovascular and other diseases.

The formation of suspensions of microbubbles In an injectable liquidcarrier suitable for echography can be produced by the release of a gasdissolved under pressure in this liquid, or by a chemical reactiongenerating gaseous products, or by admixing with the liquid soluble orinsoluble solids containing air or gas trapped or adsorbed therein.

For instance, in U.S. Pat. No. 4,446,442 (Schering), there are discloseda series of different techniques for producing suspensions of gasmicrobubbles in a sterilized injectable liquid carrier using (a) asolution of a tenside (surfactant) in a carrier liquid (aqueous) and (b)a solution of a viscosity enhancer as stabilizer. For generating thebubbles, the techniques disclosed there include forcing at high velocitya mixture of (a), (b) and air through a small aperture; or injecting (a)into (b) shortly before use together with a physiologically acceptablegas; or adding an acid to (a) and a carbonate to (b), both componentsbeing mixed together just before use and the acid reacting with thecarbonate to generate CO₂ bubbles; or adding an over-pressurized gas toa mixture of (a) and (b) under storage, said gas being released intomicrobubbles at the time when the mixture is used for injection.

EP-A-131,540 (Schering) discloses the preparation of microbubblesuspensions in which a stabilized injectable carrier liquid, e.g. aphysiological aqueous solution of salt. or a solution of a sugar likemaltose, dextrose, lactose or galactose, is mixed with solidmicroparticles (in the 0.1 to 1 μm range) of the same sugars containingentrapped air. In order to develop the suspension of bubbles in theliquid carrier, both liquid and solid components are agitated togetherunder sterile conditions for a few seconds and, once made, thesuspension must then be used immediately, i.e. it should be injectedwithin 5-10 minutes for echographic measurements; indeed, because theyare evanescent, the bubble concentration becomes too low for beingpractical after that period.

In an attempt to cure the evanescence problem. microballoons, i.e.microvesicles with a material wall, have been developed. As said before,while the microbubbles only have an immaterial or evanescent envelope,i.e. they are only surrounded by a wall of liquid whose surface tensionis being modified by the presence of a surfactant, the microballoons ormicrocapsules have a tangible envelope made of substantive material.e.g. a polymeric membrane with definite mechanical strength. In otherterms, they are microvesicles of material in which the air or gas ismore or less tightly encapsulated.

For instance, U.S. Pat. No. 4,276,885 (Tickner et al.) discloses usingsurface membrane microcapsules containing a gas for enhancing ultrasonicimages, the membrane including a multiplicity of non-toxic andnon-antigenic organic molecules. In a disclosed embodiment. thesemicrobubbles have a gelatine membrane which resists coalescence andtheir preferred size is 5-10 μm. The membrane of these microbubbles issaid to be sufficiently stable for making echographic measurements.

Air-filled microballoons without gelatin are disclosed in U.S. Pat. No.4,718,433 (Feinstein). These microvesicles are made by sonication (5 to30 kHz) of protein solutions like 5% serum albumin and have diameters inthe 2-20 μm range, mainly 2-4 μm. The microvesicles are stabilized bydenaturation of the membrane forming protein after sonication, forinstance by using heat or by chemical means, e.g. by reaction withformaldehyde or glutaraldehyde. The concentration of stablemicrovesicles obtained by this technique is said to be about 8×10⁶/ml inthe 2-4 μm range, about 10⁶/ml in the 4-5 μm range and less than 5×10⁵in the 5-6 μm range. The stability time of these microvesicles is saidto be 48 hrs or longer and they permit convenient left heart imagingafter intravenous injection. For instance, the sonicated albuminmicrobubbles when injected into a peripheral vein are capable oftranspulmonary passage. This results in echocardiographic opacificationof the left ventricle cavity as well as myocardial tissues.

Recently, still further improved microballoons for injection ultrasonicechography have been reported in EP-A-324.938 (Widder). In this documentthere are disclosed high concentrations (more than 10⁸/ml) of air-filledprotein-bounded microvesicles of less than 10 μm which have life-timesof several months or more. Aqueous suspensions of these microballoonsare produced by ultrasonic cavitation of solutions of heat denaturableproteins, e.g. human serum albumin, which operation also leads to adegree of foaming of the membrane-forming protein and its subsequenthardening by heat. Other proteins such as hemoglobin and collagen werealso said to be convenient in this process. The high storage stabilityof the suspensions of microballoons disclosed in EP-A-324.938 enablesthem to be marketed as such, i.e. with the liquid carrier phase, whichis a strong commercial asset since preparation before use is no longernecessary.

Similar advantages have been recently discovered in connection with thepreparation of aqueous microbubble suspensions, i.e. there has beendiscovered storage-stable dry pulverulent composition which willgenerate long-lasting bubble suspensions upon the addition of water.This is being disclosed in Application PCT/EP 91/00620 where liposomescomprising membrane-forming lipids are freeze-dried, and thefreeze-dried lipids, after exposure to air or a gas for a period oftime, will produce long-lasting bubble suspensions upon simple additionthereto of an aqueous liquid carrier.

Despite the many progresses achieved regarding the stability understorage of aqueous microbubble suspensions, this being either in theprecursor or final preparation stage, there still remained until now theproblem of vesicle durability when the suspensions are exposed tooverpressure, e.g. pressure variations such as that occurring afterinjection in the blood stream of a patient and consecutive to heartpulses, particularly in the left ventricle. Actually, the presentinventors have observed that, for instance in anaesthetised rabbits, thepressure variations are not sufficient to substantially alter the bubblecount for a period of time after injection. In contrast, in dogs andhuman patients, typical microbubbles or microballoons filled with commongases such as air, methane or CO₂ will collapse completely in a matterof seconds after injection due to the blood pressure effect. Thisobservation has been confirmed by others: For instance, S. GOTTLIEB etal. in J. Am. Soc. of Echocardiography 3 (1990) 238 have reported thatcross-linked albumin microballoons prepared by the sonication methodwere losing all echogenic properties after being subjected to anoverpressure of 60 Torr. It became hence Important to solve the problemand to increase the useful life of suspensions of microbubbles andmembrane bounded microballoons under pressure in order to ensure thatechographic measurements can be performed in vivo safely andreproducibly.

It should be mentioned at this stage that another category of echogenicImage enhancing agents has been proposed which resist overpressures asthey consist of plain microspheres with a porous structure, suchporosity containing air or a gas. Such microspheres are disclosed forinstance in WO-A-91/12823 (DELTA BIOTECHNOLOGY). EP-A-327 490 (SCHERING)and EP-A-458 079 (HOECHST). The drawback with the plain porousmicrospheres is that the encapsulated gas-filled free space is generallytoo small for good echogenic response and the spheres lack adequateelasticity. Hence the preference generally remains with the hollowmicrovesicles and a solution to the collapsing problem was searched.

DISCLOSURE OF THE INVENTION

This problem has now been solved by using gases or gas mixtures inconformity with the criteria outlined in the claims. Briefly, it hasbeen found that when the echogenic microvesicles are made in thepresence of a gas, respectively are filled at least in part with a gas,having physical properties in conformity with the equation below, thenthe microvesicles remarkably resist pressure >60 Torr after injectionfor a time sufficient to obtain reproducible echographic measurements:${\frac{s_{gas}}{s_{air}} \times \frac{\sqrt{{Mw}_{air}}}{\sqrt{{Mw}_{gas}}}} \leq 1$

In the foregoing equation, “s” designates the solubilities in waterexpressed as the “BUNSEN” coefficients, i.e. as volume of gas dissolvedby unit volume of water under standard conditions (1 bar. 25° C.), andunder partial pressure of the given gas of 1 atm (see the GasEncyclopaedia, Elsevier 1976). Since, under such conditions anddefinitions, the solubility of air is 0.0167, and the square root of itsaverage molecular weight (Mw) is 5.39, the above relation simplifies to:

^(s)gas/^(Mw)gas ≦0.0031

In the Examples to be found hereafter there is disclosed the testing ofechogenic microbubbles and microballoons (see the Tables) filled with anumber of different gases and mixtures thereof, and the correspondingresistance thereof to pressure increases, both in vivo and in vitro. Inthe Tables, the water solubility factors have also been taken from theaforecited Gas Encyclopaedia from “L'Air Liquide”, Elsevier Publisher(1976).

The microvesicles in aqueous suspension containing gases according tothe invention include most microbubbles and microballoons discloseduntil now for use as contrast agents for echography. The preferredmicroballoons are those disclosed in EP-A-324,938, PCT/EP91/01706 andEP-A-458 745; the preferred microbubbles are those of PCT/EP91/00620;these microbubbles are advantageously formed from an aqueous liquid anda dry powder (microvesicle precursors) containing lamellarizedfreeze-dried phospholipids and stabilizers; the microbubbles aredeveloped by agitation of this powder in admixture with the aqueousliquid carrier. The microballoons of EP-A-458 745 have a resilientinterfacially precipitated polymer membrane of controlled porosity. Theyare generally obtained from emulsions into microdroplets of polymersolutions in aqueous liquids, the polymer being subsequently caused toprecipitate from its solution to form a filmogenic membrane at thedroplet/liquid interface, which process leads to the initial formationof liquid-filled microvesicles, the liquid core thereof being eventuallysubstituted by a gas.

In order to carry out the method of the present invention, i.e. to formor fill the microvesicles, whose suspensions in aqueous carriersconstitute the desired echogenic additives, with the gases according tothe foregoing relation, one can either use, as a first embodiment, a twostep route consisting of (1) making the microvesicles from appropriatestarting materials by any suitable conventional technique in thepresence of any suitable gas, and (2) replacing this gas originally used(first gas) for preparing the microvesicles with a new gas (second gas)according to the invention (gas exchange technique).

Otherwise, according to a second embodiment, one can directly preparethe desired suspensions by suitable usual methods under an atmosphere ofthe new gas according to the invention.

If one uses the two-step route, the initial gas can be first removedfrom the vesicles (for instance by evacuation under suction) andthereafter replaced by bringing the second gas into contact with theevacuated product, or alternatively, the vesicles still containing thefirst gas can be contacted with the second gas under conditions wherethe second gas will displace the first gas from the vesicles (gassubstitution). For instance, the vesicle suspensions. or preferablyprecursors thereof (precursors here may mean the materials themicrovesicle envelopes are made of, or the materials which, uponagitation with an aqueous carrier liquid, will generate or develop theformation of microbubbles in this liquid), can be exposed to reducedpressure to evacuate the gas to be removed and then the ambient pressureis restored with the desired gas for substitution. This step can berepeated once or more times to ensure complete replacement of theoriginal gas by the new one. This embodiment applies particularly wellto precursor preparations stored dry, e.g. dry powders which willregenerate or develop the bubbles of the echogenic additive uponadmixing with an amount of carrier liquid. Hence, in one preferred casewhere microbubbles are to be formed from an aqueous phase and drylaminarized phospholipids, e.g. powders of dehydrated lyophilizedliposomes plus stabilizers, which powders are to be subsequentlydispersed under agitation in a liquid aqueous carrier phase, it isadvantageous to store this dry powder under an atmosphere of a gasselected according to the invention. A preparation of such kind willkeep indefinitely in this state and can be used at any time fordiagnosis, provided it is dispersed into sterile water before injection.

Otherwise, and this is particularly so when the gas exchange is appliedto a suspension of microvesicles in a liquid carrier phase, the latteris flushed with the second gas until the replacement (partial orcomplete) is sufficient for the desired purpose. Flushing can beeffected by bubbling from a gas pipe or, in some cases, by simplysweeping the surface of the liquid containing the vesicles under gentleagitation with a stream (continuous or discontinuous) of the new gas. Inthis case, the replacement gas can be added only once in the flaskcontaining the suspension and allowed to stand as such for a while, orit can be renewed one or more times in order to assure that the degreeof renewal (gas exchange) is more or less complete.

Alternatively, in a second embodiment as said before, one will effectthe full preparation of the suspension of the echogenic additivesstarting with the usual precursors thereof (starting materials), asrecited in the prior art and operating according to usual means of saidprior art, but in the presence of the desired gases or mixture of gasesaccording to the invention instead of that of the prior art whichusually recites gases such as air, nitrogen, CO₂ and the like.

The physiologically acceptable gas is selected from CF₄, CBrF₃, CClF₃,CCl₂F₂, CBrClF₂, C₂Cl₂F₄, C₂ClF₅, C₂F₆, C₄F₈ and C₄F₁₀.

It should be noted that in general the preparation mode involving onefirst type of gas for preparing the microvesicles and, thereafter,substituting the original gas by a second kind of gas, the latter beingintended to confer different echogenic properties to said microvesicles,has the following advantage: As will be best seen from the results inthe Examples hereinafter, the nature of the gas used for making themicrovesicles, particularly the microballoons with a polymer envelope,has a definitive influence on the overall size (i.e. the average meandiameter) of said microvesicles; for instance, the size of microballoonsprepared under air with precisely set conditions can be accuratelycontrolled to fall within a desired range, e.g. the 1 to 10 μm rangesuitable for echographying the left and right heart ventricles. This notso easy with other gases, particularly the gases in conformity with therequirements of the present invention; hence, when one wishes to obtainmicrovesicles in a given size range but filled with gases the nature ofwhich would render the direct preparation impossible or very hard, onewill much advantageously rely on the two-steps preparation route, i.e.one will first prepare the microvesicles with a gas allowing moreaccurate diameter and count control, and thereafter replace the firstgas by a second gas by gas exchange.

In the description of the Experimental part that follows (Examples),gas-filled microvesicles suspended in water or other aqueous solutionshave been subjected to pressures over that of ambient. It was noted thatwhen the overpressure reached a certain value (which is generallytypical for a set of microsphere parameters and working conditions liketemperature, compression rate, nature of carrier liquid and its contentof dissolved gas (the relative importance of this parameter will bedetailed hereinafter), nature of gas filler, type of echogenic material,etc.), the microvesicles started to collapse, the bubble countprogressively decreasing with further increasing the pressure until acomplete disappearance of the sound reflector effect occurred. Thisphenomenon was better followed optically, (nephelometric measurements)since it Is paralleled by a corresponding change in optical density.i.e. the transparency of the medium increases as the bubbleprogressively collapse. For this the aqueous suspension of microvesicles(or an appropriate dilution thereof) was placed in a spectrophotometriccell maintained at 25° C. (standard conditions) and the absorbance wasmeasured continuously at 600 or 700 nm, while a positive hydrostaticoverpressure was applied and gradually increased. The pressure wasgenerated by means of a peristaltic pump (GILSON's Mini-puls) feeding avariable height liquid column connected to the spectrophotometric cell,the latter being sealed leak-proof. The pressure was measured with amercury manometer calibrated in Torr. The compression rate with time wasfound to be linearly correlated with the pump's speed (rpm's). Theabsorbance in the foregoing range was found to be proportional to themicrovesicle concentration in the carrier liquid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph which relates the bubble concentration (bubble count),expressed in terms of optical density in the aforementioned range, andthe pressure applied over the bubble suspension. The data for preparingthe graph are taken from the experiments reported in Example 4.

FIG. 1 shows graphically that the change of absorbance versus pressureis represented by a sigmoid-shaped curve. Up to a certain pressurevalue, the curve is nearly flat which indicates that the bubbles arestable. Then, a relatively fast absorbance drop occurs, which indicatesthe existence of a relatively narrow critical region within which anypressure increase has a rather dramatic effect on the bubble count. Whenall the microvesicles have disappeared, the curve levels off again. Acritical point on this curve was selected in the middle between thehigher and lower optical readings, i.e. intermediate between the“full”-bubble (OD max) and the “no”-bubble (OD min) measurements, thisactually corresponding where about 50% of the bubbles initially presenthave disappeared, i.e. where the optical density reading is about halfthe initial reading, this being set, in the graph, relative to theheight at which the transparency of the pressurized suspension ismaximal (base line). This point which is also in the vicinity where theslope of the curve is maximal is defined as the critical pressure PC. Itwas found that for a given gas, PC does not only depend on theaforementioned parameters but also, and particularly so, on the actualconcentration of gas (or gases) already dissolved in the carrier liquid:the higher the gas concentration, the higher the critical pressure. Inthis connection, one can therefore increase the resistance to collapseunder pressure of the microvesicles by making the carrier phasesaturated with a soluble gas. the latter being the same, or not, (i.e. adifferent gas) as the one that fills the vesicles. As an example,air-filled microvesicles could be made very resistant to overpressures(>120 Torr) by using, as a carrier liquid, a saturated solution of CO₂.Unfortunately, this finding is of limited value in the diagnostic fieldsince once the contrast agent is injected to the bloodstream of patients(the gas content of which is of course outside control), it becomesdiluted therein to such an extent that the effect of the gas originallydissolved in the injected sample becomes negligible.

Another readily accessible parameter to reproducibly compare theperformance of various gases as microsphere fillers is the width of thepressure interval (ΔP) limited by the pressure values under which thebubble counts (as expressed by the optical densities) is equal to the75% and 25% of the original bubble count. Now, it has been surprisinglyfound that for gases where the pressure difference DP=P₂₅−P₇₅- exceeds avalue of about 25-30 Torr, the killing effect of the blood pressure onthe gas-filled microvesicles is minimized, i.e. the actual decrease inthe bubble count is sufficiently slow not to impair the significance,accuracy and reproducibility of echographic measurements.

It was found. in addition, that the values of PC and ΔP also depend onthe rate of rising the pressure in the test experiments illustrated byFIG. 1, i.e. in a certain interval of pressure increase rates (e.g. inthe range of several tens to several hundreds of Torr/min), the higherthe rate, the larger the values for PC and ΔP. For this reason, thecomparisons effected under standard temperature conditions were alsocarried out at the constant increase rate of 100 Torr/min. It shouldhowever be noted that this effect of the pressure increase rate on themeasure of the PC and ΔP values levels off for very high rates; forinstance the values measured under rates of several hundreds of Torr/minare not significantly different from those measured under conditionsruled by heart beats.

Although the very reasons why certain gases obey the aforementionedproperties, while others do not, have not been entirely clarified, itwould appear that some relation possibly exists in which, in addition tomolecular weight and water solubility, dissolution kinetics, and perhapsother parameters, are involved. However these parameters need not beknown to practise the present invention since gas eligibility can beeasily determined according to the aforediscussed criteria.

The gaseous species which particularly suit the invention are, forinstance, halogenated hydrocarbons like the freons and stablefluorinated chalcogenides like SF₆, SeF₆ and the like.

It has been mentioned above that the degree of gas saturation of theliquid used as carrier for the microvesicles according to the inventionhas an importance on the vesicle stability under pressure variations.Indeed, when the carrier liquid in which the microvesicles are dispersedfor making the echogenic suspensions of the invention is saturated atequilibrium with a gas, preferably the same gas with which themicrovesicles are filled, the resistance of the microvesicles tocollapse under variations of pressure is markedly increased. Thus, whenthe product to be used as a contrast agent is sold dry to be mixed justbefore use with the carrier liquid (see for instance the productsdisclosed In PCT/EP91/00620 mentioned hereinbefore), it is quiteadvantageous to use, for the dispersion, a gas saturated aqueouscarrier. Alternatively, when marketing ready-to-use microvesiclesuspensions as contrast agents for echography, one will advantageouslyuse as the carrier liquid for the preparation a gas saturated aqueoussolution; in this case the storage life of the suspension will beconsiderably increased and the product may be kept substantiallyunchanged (no substantial bubble count variation) for extended periods,for instance several weeks to several months, and even over a year inspecial cases. Saturation of the liquid with a gas may be effected mosteasily by simply bubbling the gas into the liquid for a period of timeat room temperature.

EXAMPLE 1

Albumin microvesicles filled with air or various gases were prepared asdescribed in EP-A-324 938 using a 10 ml calibrated syringe filled with a5% human serum albumin (HSA) obtained from the Blood TransfusionService, Red-Cross Organization, Bern, Switzerland. A sonicator probe(Sonifier Model 250 from Branson Ultrasonic Corp, USA) was lowered intothe solution down to the 4 ml mark of the syringe and sonication waseffected for 25 sec (energy setting=8). Then the sonicator probe wasraised above the solution level up to the 6 ml mark and sonication wasresumed under the pulse mode (cycle=0.3) for 40 sec. After standingovernight at 4° C., a top layer containing most of the microvesicles hadformed by buoyancy and the bottom layer containing unused albumin debrisof denatured protein and other insolubles was discarded. Afterresuspending the microvesicles in fresh albumin solution the mixture wasallowed to settle again at room temperature and the upper layer wasfinally collected. When the foregoing sequences were carried out underthe ambient atmosphere, air filled microballoons were obtained. Forobtaining microballoons filled with other gases, the albumin solutionwas first purged with a new gas, then the foregoing operationalsequences were effected under a stream of this gas flowing on thesurface of the solution; then at the end of the operations, thesuspension was placed in a glass bottle which was extensively purgedwith the desired gas before sealing.

The various suspensions of microballoons filled with different gaseswere diluted to 1:10 with distilled water saturated at equilibrium withair, then they were placed in an optical cell as described above and theabsorbance was recorded while increasing steadily the pressure over thesuspension. During the measurements, the suspensions temperature waskept at 25° C.

The results are shown in the Table 1 below and are expressed in terms ofthe critical pressure PC values registered for a series of gases definedby names or formulae, the characteristic parameters of such gases, i.e.Mw and water solubility being given, as well as the original bubblecount and bubble average size (mean diameter in volume).

TABLE 1 Bubble Bubble Solu- count size PC S_(gas)/ Sample Gas Mw bility(10⁸/ml) (μm) (Torr) Mw AFre1 CF₄ 88 .0038 0.8 5.1 120 .0004 AFre2 CBrF₃149 .0045 0.1 11.1 104 .0004 ASF1 SF₆ 146 .005  13.9 6.2 150 .0004 ASF2SF₆ 146 .005  2.0 7.9 140 .0004 AN1 N₂ 28 .0144 0.4 7.8 62 .0027 A14 Air29 .0167 3.1 11.9 53 .0031 A18 Air 29 .0167 3.8 9.2 52 — A19 Air 29.0167 1.9 9.5 51 — AMe1 CH₄ 16 .032  0.25 8.2 34 .008  AKrl Kr 84 .059 0.02 9.2 86 .006  AX1 Xe 131 .108  0.06 17.2 65 .009  AX2 Xe 131 .108 0.03 16.5 89 .009 

From the results of Table 1, it is seen that the critical pressure PCincreases for gases of lower solubility and higher molecular weight. Itcan therefore be expected that microvesicles filled with such gases willprovide more durable echogenic signals in vivo. It can also be seen thataverage bubble size generally increases with gas solubility.

EXAMPLE 2

Aliquots (1 ml) of some of the microballoon suspensions prepared inExample 1 were injected in the jugular vein of experimental rabbits inorder to test echogenicity in vivo. Imaging of the left and right heartventricles was carried out in the grey scale mode using an Acuson128-XP5 echography apparatus and a 7.5 MHz transducer. The duration ofcontrast enhancement in the left ventricle was determined by recordingthe signal for a period of time. The results are gathered in Table 2below which also shows the PC of the gases used.

TABLE 2 Sample Duration of PC (Gas) contrast (sec) (Torr) AMe1 (CH₄)zero 34 A14 (air) 10 53 A18 (air) 11 52 AX1 (Xe) 20 65 AX2 (Xe) 30 89ASF2 (SF₆) >60   140 

From the above results, one can see the existence of a definitecorrelation between the critical pressure of the gases tried and thepersistence in time of the echogenic signal.

EXAMPLE 3

A suspension of echogenic air-filled galactose microparticles (Echovist®from SCHERING AG) was obtained by shaking for 5 sec 3 g of the solidmicroparticles in 8.5 ml of a 20% galactose solution. In otherpreparations, the air above a portion of Echovist® particles wasevacuated (0.2 Torr) and replaced by an SF₆ atmosphere, whereby, afteraddition of the 20% galactose solution, a suspension of microparticlescontaining associated sulfur hexafluoride was obtained. Aliquots (1 ml)of the suspensions were administered to experimental rabbits (byinjection in the jugular vein) and imaging of the heart was effected asdescribed in the previous example. In this case the echogenicmicroparticles do not transit through the lung capillaries, henceimaging is restricted to the right ventricle and the overall signalpersistence has no particular significance. The results of Table 3 belowshow the value of signal peak intensity a few seconds after injection.

TABLE 3 Sample Signal peak No Gas (arbitrary units) Gal1 air 114 Gal2air 108 Gal3 SF₆ 131 Gal4 SF₆ 140

It can be seen that sulfur hexafluoride, an inert gas with low watersolubility, provides echogenic suspensions which generate echogenicsignals stronger than comparable suspensions filled with air. Theseresults are particularly interesting in view of the teachings ofEP-A-441 468 and 357 163 (SCHERING) which disclose the use forechography purposes of micropartcles, respectively, cavitate andclathrate compounds filled with various gases including SF6; thesedocuments do not however report particular advantages of SF6 over othermore common gases with regard to the echogenic response.

EXAMPLE 4

A series of echogenic suspensions of gas-filled microbubbles wereprepared by the general method set forth below:

One gram of a mixture of hydrogenated soya lecithin (from NattermannPhospholipids GmbH, Germany) and dicetyl-phosphate (DCP), in 9/1 molarratio, was dissolved in 50 ml of chloroform. and the solution was placedin a 100 ml round flask and evaporated to dryness on a Rotavaporapparatus. Then, 20 ml of distilled water were added and the mixture wasslowly agitated at 75° C. for an hour. This resulted in the formation ofa suspension of multilamellar liposomes (MLV) which was thereafterextruded at 75° C. through, successively, 3 μm and 0.8 μm polycarbonatemembranes (Nuclepore®). After cooling, 1 ml aliquots of the extrudedsuspension were diluted with 9 ml of a concentrated lactose solution (83g/l), and the diluted suspensions were frozen at −45° C. The frozensamples were thereafter freeze-dried under high vacuum to a free-flowingpowder in a vessel which was ultimately filled with air or a gas takenfrom a selection of gases as indicated in Table 4 below. The powderysamples were then resuspended in 10 ml of water as the carrier liquid,this being effected under a stream of the same gas used to fill the saidvessels. Suspension was effected by vigorously shaking for 1 min on avortex mixer.

The various suspensions were diluted 1:20 with distilled waterequilibrated beforehand with air at 25° C. and the dilutions were thenpressure tested at 25° C. as disclosed in Example 1 by measuring theoptical density in a spectrophotometric cell which was subjected to aprogressively increasing hydrostatic pressure until all bubbles hadcollapsed. The results are collected in Table 4 below which, in additionto the critical pressure PC, gives also the ΔP values (see FIG. 1).

TABLE 4 Bubble Sample Solubility count PC ΔP No Gas M_(w) in H₂O(10⁸/ml) (Torr) (Torr) LFre1 CF₄ 88 .0038 1.2 97 35 LFre2 CBrF₃ 149.0045 0.9 116  64 LSF1 SF₆ 146 .005  1.2 92 58 LFre3 C₄F₈ 200 .016  1.5136  145  L1 air 29 .0167 15.5 68 17 L2 air 29 .0167 11.2 63 17 LAr1 Ar40 .031  14.5 71 18 LKr1 Kr 84 .059  12.2 86 18 LXe1 Xe 131 .108  10.192 23 LFre4 CHClF₂ 86 .78  — 83 25

The foregoing results clearly indicate that the highest resistance topressure increases is provided by the most water-insoluble gases. Thebehavior of the microbubbles is therefore similar to that of themicroballoons in this regard. Also, the less water-soluble gases withthe higher molecular weights provide the flattestbubble-collapse/pressure curves (i.e. ΔP is the widest) which is also animportant factor of echogenic response durability in vivo, as indicatedhereinbefore.

EXAMPLE 5

Some of the microbubble suspensions of Example 4 were injected to thejugular vein of experimental rabbits as indicated in Example 2 andimaging of the left heart ventricle was effected as indicatedpreviously. The duration of the period for which a useful echogenicsignal was detected was recorded and the results are shown in Table 5below in which C₄F₈ indicates octafluorocyclobutane.

TABLE 5 Contrast duration Sample No Type of gas (sec) L1 Air 38 L2 Air29 LMe1 CH₄ 47 LKr1 Krypton 37 LFre1 CF₄ >120 LFre2 CBrF₃ 92 LSF1SF₆ >112 LFre3 C₄F₈ >120

These results indicate that, again in the case of microbubbles, thegases according to the criteria of the present invention will provideultrasonic echo signal for a much longer period than most gases useduntil now.

EXAMPLE 6

Suspensions of microbubbles were prepared using different gases exactlyas described in Example 4, but replacing the lecithin phospholipidingredient by a mole equivalent of diarachidoylphosphatidylcholine (C₂₀fatty acid residue) available from Avanti Polar Lipids, Birmingham,Ala., USA. The phospholipid to DCP molar ratio was still 9/1. Then thesuspensions were pressure tested as in Example 4; the results, collectedin Table 6A below, are to be compared with those of Table 4.

TABLE 6A Solu- Bubble Sample Type of Mw of bility count PC ΔP No gas gasin Water (10⁸/ml) (Torr) (Torr) LFre1 CF₄ 88 .0038 3.4 251 124 LFre2CBrF₃ 149 .0045 0.7 121 74 LSF1 SF₆ 146 .005  3.1 347 >150 LFre3 C₄F₈200 .016  1.7 >350 >200 L1 Air 29 .0167 3.8 60 22 LBu1 Butane 58 .027 0.4 64 26 LAr1 Argon 40 .031  3.3 84 47 LMe1 CH₄ 16 .032  3.0 51 19 LEt1C₂H₆ 44 .034  1.4 61 26 LKr1 Kr 84 .059  2.7 63 18 LXe1 Xe 131 .108  1.460 28 LFre4 CHClF₂ 86 .78  0.4 58 28

The above results, compared to that of Table 4, show that, at least withlow solubility gases, by lengthening the chain of the phospholipid fattyacid residues, one can dramatically increase the stability of theechogenic suspension toward pressure increases. This was furtherconfirmed by repeating the foregoing experiments but replacing thephospholipid component by its higher homolog, i.e.di-behenoyl-phosphatidylcholine (C₂₂ fatty acid residue). In this case,the resistance to collapse with pressure of the microbubbles suspensionswas still further increased.

Some of the microbubbles suspensions of this Example were tested in dogsas described previously for rabbits (imaging of the heart ventriclesafter injection of 5 ml samples in the anterior cephalic vein). Asignificant enhancement of the useful in-vivo echogenic response wasnoted. in comparison with the behavior of the preparations disclosed inExample 4, i.e. the increase in chain length of the fatty-acid residuein the phospholipid component increases the useful life of the echogenicagent in-vivo.

In the next Table below, there is shown the relative stability in theleft ventricle of the rabbit of microbubbles (SF₆) prepared fromsuspensions of a series of phospholipids whose fatty acid residues havedifferent chain lengths (<injected dose: 1 ml/rabbit).

TABLE 6B Chain Duration of Phospho- length PC ΔP contrast lipld (C_(n))(Torr) (Torr) (sec) DMPC 14  57 37 31 DPPC 16 100 76 105 DSPC 18 115 95120 DAPC 20 266 190  >300

It has been mentioned hereinabove that for the measurement of resistanceto pressure described in these Examples, a constant rate of pressurerise of 100 Torr/min was maintained. This is justified by the resultsgiven below which show the variations of the PC values for differentgases in function to the rate of pressure increase. In these samplesDMPC was the phospholipid used.

PC (Torr) Gas Rate of pressure increase (Torr/min) sample 40 100 200 SF₆51 57 82 Air 39 50 62 CH₄ 47 61 69 Xe 38 43 51 Freon 22 37 54 67

EXAMPLE 7

A series of albumin microballoons as suspensions in water were preparedunder air in a controlled sphere size fashion using the directions givenin Example 1. Then the air in some of the samples was replaced by othergases by the gas-exchange sweep method at ambient pressure. Then, afterdiluting to 1:10 with distilled water as usual, the samples weresubjected to pressure testing as in Example 1. From the results gatheredin Table 7 below, it can be seen that the two-steps preparation modegives, in some cases, echo-generating agents with better resistance topressure than the one-step preparation mode of Example 1.

TABLE 7 Initial bubble Sample Type of Mw of Solubility count PC No gasthe gas in water (10⁸/ml) (Torr) A14 Air 29 .0167 3.1 53 A18 Air 29.0167 3.8 52 A18/SF₆ SF₆ 146  .005  0.8 115  A18/C₂H₆ C₂H₆ 30 .042  3.472 A19 Air 29 .0167 1.9 51 A19/SF₆ SF₆ 146  .005  0.6 140  A19/Xe Xe131  .108  1.3 67 A22/CF₄ CF₄ 88 .0038 1.0 167  A22/Kr Kr 84 .059  0.685

EXAMPLE 8

The method of the present invention was applied to an experiment asdisclosed in the prior art, for instance Example 1 WO-92/11873. Threegrams of Pluronic® F68 (a copolymer of polyoxyethylene-polyoxypropylenewith a molecular weight of 8400), 1 g of dipalmitoylphosphatidylglycerol(Na salt, AVANTI Polar Lipids) and 3.6 g of glycerol were added to 80 mlof distilled water. After heating at about 80° C., a clear homogenoussolution was obtained. The tenside solution was cooled to roomtemperature and the volume was adjusted to 100 ml. In some experiments(see Table 8) dipalmitoylphosphatidylglycerol was replaced by a mixtureof diarachidoylphosphatidylcholine (920 mg) and 80 mg ofdipalmitoylphosphatidic acid (Na salt, AVANTI Polar lipids).

The bubble suspensions were obtained by using two syringes connected viaa three-way valve. One of the syringes was filled with 5 ml of thetenside solution while the other was filled with 0.5 ml of air or gas.The three-way valve was filled with the tenside solution before it wasconnected to the gas-containing syringe. By alternatively operating thetwo pistons, the tenside solutions were transferred back and forthbetween the two syringes (5 times in each direction), milky suspensionswere formed. After dilution (1:10 to 1:50) with distilled watersaturated at equilibrium with air, the resistance to pressure of thepreparations was determined according to Example 1. The pressureincrease rate was 240 Torr/min. The following results were obtained:

TABLE 8 Phospholipid Gas Pc (mm Hg) DP (mm Hg) DPPG air  28  17 DPPG SF₆138 134 DAPC/DPPA 9/1 air  46  30 DAPC/DPPA 9/1 SF₆ 269 253

It follows that by using the method of the invention and replacing airwith other gases e.g. SF₆ even with known preparations a considerableimprovements i.e. increase in the resistance to pressure may beachieved. This is true both in the case of negatively chargedphospholipids (e.g. DPPG) and in the case of mixtures of neutral andnegatively charged phospholipids (DAPC/DPPA).

The above experiment further demonstrates that the recognised problemsensitivity of microbubbles and microballoons to collapse when exposedto pressure i.e. when suspensions are injected into living beings, hasadvantageously been solved by the method of the invention. Suspensionswith microbubbles or microballoons with greater resistance againstcollapse and greater stability can advantageously be produced providingsuspensions with better reproducibility and improved safety ofechographic measurements performed in vivo on a human or animal body.

The invention described herein can be further elucidated by thedescription of the following representative (but not limiting)embodiments, numbered 1-18:

1. A method for imparting resistance against collapsing to contrastagents for ultrasonic echography which consist of gas-filledmicrovesicles in suspension in aqueous liquid carrier phases, i.e.,either microbubbles bounded by an evanescent gas/liquid interfacialclosed surface, or microballoons bounded by a material envelope, saidcollapsing resulting, at least in part, from pressure increaseseffective, e.g., when the said suspensions are injected into the bloodstream of patients, said method comprising forming said microvesicles inthe presence of a gas, or if the microvesicles are already made fillingthem with this gas, which is a physiologically acceptable gas, or gasmixture, at least a fraction of which has a solubility in waterexpressed in liters of gas by liter of water under standard conditionsdivided by the square root of the molecular weight in daltons which doesnot exceed 0.003.

2. The method of embodiment 1, which is carried out in two steps, in thefirst step the microvesicles or dry precursors thereof are initiallyprepared under an atmosphere of a first gas, then in the second step atleast a fraction of the first gas is substantially substituted by asecond gas, the latter being said physiologically acceptable gas.

3. The method of embodiment 1, in which the physiologically acceptablegas used is selected from SF₆ or Freon® such as CF₄, CBrF₃, C₄F₈, CClF₃,C₂F₆, C₂ClF₅, CBrClF₂, C₂Cl₂F₄, and C₄F₁₀.

4. The method of embodiment 2, in which the gas used in the first stepis a kind that allows effective control of the average size andconcentration of the microvesicles in the carrier liquid, and thephysiologically acceptable gas added in the second step ensuresprolonged useful echogenic life to the suspension for in vivo ultrasonicimaging.

5. The method of embodiment 1, in which the aqueous phase carrying themicrobubbles contains dissolved film-forming surfactants in lamellar orlaminar form, said surfactants stabilizing the microbubbles boundary atthe gas to liquid interface.

6. The method of embodiment 5, in which said surfactants comprise one ormore phospholipids.

7. The method of embodiment 6, in which at least part of thephospholipids are in the form of liposomes.

8. The method of embodiment 6, in which at least one of thephospholipids is a diacylphosphatidyl compound wherein the acyl group isa C₁₆ fatty acid residue or a higher homologue thereof.

9. The method of embodiments 1 and 2, in which the microballoon materialenvelope is made of an organic polymeric membrane.

10. The method of embodiment 9, in which the polymers of the membraneare selected from polylactic or polyglycolic acid and their copolymers,reticulated serum albumin, reticulated haemoglobin, polystyrene, andesters of polyglutamic and polyaspartic acids.

11. The method of embodiment 1, in which the forming of themicrovesicles with said physiologically acceptable gas is effected byalternately subjecting dry precursors thereof to reduced pressure andrestoring the pressure with said gas, and finally dispersing theprecursors in a liquid carrier.

12. The method of embodiment 1, in which the filling of themicroballoons with said physiologically acceptable gas is effected bysimply flushing the suspension with said gas under ambient pressure.

13. The method of embodiment 1, which comprises making the microvesiclesby any standard method known in the art but operating under anatmosphere composed at least in part of said gas.

14. Suspensions of gas filled microvesicles distributed in an aqueouscarrier liquid to be used as contrast agents in ultrasonic echography,characterized in that the gas is physiologically acceptable and suchthat at least a portion thereof has a solubility in water, expressed inliter of gas by liter of water under standard conditions, divided by thesquare root of the molecular weight which does not exceed 0.003.

15. The aqueous suspensions of embodiment 14, characterized in that thegas is such that the pressure difference ΔP between those pressureswhich, when applied under standard conditions and at a rate of about 100Torr/min to the suspension cause the collapsing of about 75%,respectively 25%, of the microvesicles initially present, is at least 25Torr.

16. Aqueous suspensions according to embodiment 14, in which themicrovesicles are microbubbles filled with said physiologicallyacceptable gas suspended in an aqueous carrier liquid containingphospholipids whose fatty acid residues contain 16 carbons or more.

17. Contrast agents for echography in precursor form consisting of a drypowder comprising lyophilized liposomes and stabilizers, this powderbeing dispersible in aqueous liquid carriers to form echogenicsuspensions of gas-filled microbubbles, characterized in that it isstored under an atmosphere comprising a physiologically acceptable gaswhose solubility in water, expressed in liter of gas by liter of waterunder standard conditions, divided by the square root of the molecularweight does not exceed 0.003.

18. The contrast agent precursors of embodiment 17, in which theliposomes comprise phospholipids whose fatty acid residues have 16 ormore carbon atoms.

What is claimed is:
 1. A contrast agent for ultrasonic echography havingan improved resistance to pressure variations comprising, as asuspension in an aqueous carrier liquid, microballoons consisting of afreon gas bounded by an organic polymer envelope made from denaturedserum albumin.
 2. A contrast agent for ultrasonic echography having animproved resistance to pressure variations comprising, as a suspensionin an aqueous carrier liquid, microballoons consisting of a freon gasbubble encapsulated by an organic polymer envelope made from denaturedserum albumin.
 3. A contrast agent for ultrasonic echography having animproved resistance to pressure variations comprising, as a suspensionin an aqueous carrier liquid, microballoons comprising a freon gasbubble encapsulated by an organic polymer envelope made from denaturedserum albumin, said envelope being in contact only with the gas and theaqueous carrier liquid.
 4. A contrast agent for ultrasonic echographyhaving resistance to pressure variations comprising: a) a plurality ofpolymeric structures, each polymeric structure consisting of denaturedserum albumin in a hollow microballoon form, b) a freon gas that iscontained within the hollow microballoon form, and c) an aqueouspharmaceutical formulation for injection.
 5. A contrast agent forultrasonic echography having resistance to pressure variationscomprising: a) a plurality of polymeric structures, each polymericstructure consisting of denatured serum albumin, b) a freon gas that iscontained within the polymeric structure, and c) an aqueouspharmaceutical formulation for injection.
 6. A contrast agent forultrasonic echography comprising, as a suspension in an aqueous carrierliquid, microballoons consisting of a gas comprising a halogenatedhydrocarbon bounded by an organic polymer envelope made from denaturedserum albumin.
 7. A contrast agent for ultrasonic echography comprising,as a suspension in an aqueous carrier liquid, microballoons consistingof a gas bubble comprising a halogenated hydrocarbon encapsulated by anorganic polymer envelope made from denatured serum albumin.
 8. Acontrast agent for ultrasonic echography comprising, as a suspension inan aqueous carrier liquid, microballoons consisting of a gas bubblecomprising a halogenated hydrocarbon encapsulated by an organic polymerenvelope made from denatured serum albumin, said envelope being incontact only with the gas and the aqueous carrier liquid.
 9. A contrastagent for ultrasonic echography having resistance to pressure variationscomprising: a) a plurality of polymeric structures, each polymericstructure consisting of denatured serum albumin in a hollow microballoonform, b) gas comprising a halogenated hydrocarbon that is containedwithin the hollow microballoon form, and c) an aqueous pharmaceuticalformulation for injection.
 10. A contrast agent for ultrasonicechography comprising: a) a plurality of polymeric structures, eachpolymeric structure consisting of denatured serum albumin, b) gascomprising a halogenated hydrocarbon that is contained within thepolymeric structure, and c) an aqueous pharmaceutical formulation forinjection.